Construction And High Level Expression Of The Fusion Gene Of Streptococcus Pneumoniae PspA Subnit And Ply Subnit

The preventive effect of Streptococcus pneumoniae vaccines are becoming more and more important as the hazards of Streptococcus pneumoniae as large and the growing number of resistant strains of Streptococcus pneumoniae vaccine. Pneumococcal surface protein A (PspA) and pneumolysin have been identified as candidate pneumococcal vaccine factors, although the focus of their immune protection varies. The immune protective effect of PspA mainly is anti-colonization of S. pn, Ply is intracellular toxin of S. pn, the immune protection must take palce after the antolyzation of S. pn. For mice,Wild Ply has an immune protection effect. To human, it is not appropriate because of cytotoxic. Mutate the gene complement activation necessary and cytotoxicity fragement ofΔA146R147 to no toxic effects "pneumolysin". It was founded that in mice the immunize cytotoxicity can reduce 99.5%, so it can be widely used as a carrier protein fusion vaccine.This study intends to combine the advantages of them. Construct the protokaryon expression vector containing PspA and Ply genes using molecular biology gene engineering methods in erder to investgate the effect of these two candidate factors in S.pn protein vaccine at the same time.During the Follow-up experiment, the biological activity of recombinant fusion protein will be identified by animal experiments and in vitro cell adhesion inhibition tests, in order to observe the protective effect of immunization of animals and against Streptococcus pneumoniae in inhibition of host cell adhesion, so as to explore the potential value of the fusion protein vaccine of Streptococcus pneumoniae in the future. Objective:To amplify the mutated gene of Ply using the site-directed mutagenesis technigues and construct the protokaryon expression vector containing PspA andΔPly genes using molecular biology gene engineering methods.Methods:1.To design the primers of pnemococcal surface protein A and pneumolysin according to the gene sequence of Streptococcus pneumoniae TIGR4. The mutanted Ply gene was obtained by the site-directed mutagenesis of pneumolysin and PspA by PCR techniques.2.There fragments were linked to construct the prokaryotic expression vector pET32a-PspA-ΔPly, which was identified by endonuclease digestion and DNA sequencing.3.The reassembled plasmid was then transferred into E. coli BL21(DE3) and expressed through induced expression for different time, and at different temperature and concentration, which was identified by Western blot.Results:1.Successful construction of ply mutant gene in AA146R147 base pair by sequence.2.Except the mutant site, the result of DNA sequence analysis showed that the cloned PspA-ΔPly gene sequence was completely corresponding to Gene Bank data.3.SDS-PAGE and Western Blotting showed that the expressed PspA-ΔPly fusion protein was about 120×103.Conclusions:Recombinant plasmids of pET32a-PspA-ΔPly was constructed successfully, Which lays the foundation of the research of a new kind of anti-Streptococcus pneumoniae protein vaccine.